1. Field of the Invention
The present invention relates to a process for the preparation of riboflavin by fermentation.
Riboflavin can be prepared in a high yield by the fermentation method according to the present invention, using acetic acid as carbon source. The present invention further relates to a process for obtaining riboflavin formed by fermentation as crystals from the culture. Riboflavin is of value for the applications as medicine and food-stuff additives or colorants for food.
2. Description of the Prior Art
Typical riboflavin-producing microorganisms known at present are Ascomycetes such as Ashbya gossypii or Eremothecium ashbyii, with which riboflavin for food-stuff is industrially prepared from saccharides for the production of additives for food-stuff.
It is known that riboflavin is produced not only by Ascomycetes but also by some bacteria belonging to the genus Clostridium or yeasts of the genus Candida, Saccharomyces, or Hansenula (see Progress in Industrial Microbiology, Vol. 1, p. 139 (1959)). However, the productivity of riboflavin is low when bacteria or yeasts are used and it is also noticed that the presence of iron ions in trace amounts greatly decreases the productivity. Because of these disadvantages, the production of riboflavin with bacteria or yeasts has not yet been put into industrial practice (see Annual Review of Microbiology, Vol. 26, p. 369 (1972)).
Some of the present inventors reported on a process for the preparation of riboflavin by fermentation using acetic acid as a carbon source (see Takao, Agr. Biol. Chem, Vol. 28, pp. 559, 566 & 765 (1964)).
As to the recovery of riboflavin out of the culture, riboflavin for food-stuff additives is obtained by drying the entire culture without isolation of riboflavin. In that case, a method of utilizing the difference in specific gravity is suggested in order to obtain as high a riboflavin concentration as possible (see Japanese Patent Laid-Open No. 159800/1980).
Highly purified crystalline riboflavin, which can be used for medicaments and the like, has been obtained by heating the culture to solubilize riboflavin, separating insoluble matters comprising microorganisms from the solution, and thereafter separating riboflavin from that solution by temporarily changing riboflavin into a less soluble form (Economic Microbiology, Vo. 2, p. 315, Academic Press).
An example of this method is described in detail in Japan Patent Publication No. 10155/1978, according to which riboflavin is converted into reduced-form riboflavin having low solubility by the addition of a hydrosulfite and precipitated as crude crystals. The obtained crystals are oxidized in an acidic suspension and purified by recrystallization. Purified riboflavin can be prepared by this method, but the yields were too low for practical use. According to Example 1 of Japan Patent Publication No. 13276/1982, the heated culture solution from which bacteria have been removed is concentrated and reduced with titanium trichloride to form a precipitate. The precipitate is oxidized in air and purified by dissolution in hydrochloric acid and alkali precipitation (29% yield after purification). It is also known to extract the product using butanol or other organic solvents in the treatment of reduced-form (leuco) riboflavin (U.S. Pat. No. 2,464,243).